2H-1-benzopyran derivatives processes for their preparation and pharmaceutical compositions thereof

ABSTRACT

2H-1-Benzopyran derivatives, processes for their preparation and use thereof for the preparation of pharmaceutical compositions for the prevention and treatment of postmenopausal pathologies.

[0001] The present invention relates to 2H-1-benzopyran derivatives,processes for their preparation and their pharmaceutical compositions.

[0002] More precisely, the invention relates to 2H-1-benzopyranderivatives of general formula:

[0003] wherein R₁ and R₂ are independently H, alkyl, haloalkyl, alkenylor haloalkenyl, or together with nitrogen atom they are bound to, canform a 4- to 8-membered heterocyclic ring

[0004] X is H, alkyl, aryl, nitro, halo, O—R₃ wherein R₃ is H, alkyl,aryl, alkanoyl, aryloyl

[0005] X₁ is H, alkyl or alkoxy and when X and X₁ are alkyl they canform together with the carbon atom they are bound to, a fused aromaticring to give an α-naphthalenyl

[0006] Y is H, alkyl, alkanoyl, aryloyl, alkylaminocarbonyl,alkyloxycarbonyl

[0007] Z is H, O—R₄ wherein R₄ is H, alkyl, alkanoyl, aryloyl

[0008] m is 1,2

[0009] n is 0,1

[0010] p is 2-6

[0011]

represents a single or double bond between the atoms in positions 3-4 or4-4a: when n is 1 the double bond may be alternatively exocyclic orendocyclic to give respectively a 4-benzylidenechroman for m=1 or a4-benzyl-chrom-3-ene for m=2; when n is 0 the endocyclic bond 3-4 may bea single or a double bond and when n is 0 and the endocyclic bond 3-4 isa single bond, Z is not H.

[0012] The compounds of the invention can exist in the form of salts.

[0013] The compounds bearing basic groups can exist in the form oforganic or inorganic acid addition salts.

[0014] The compounds bearing acid groups can exist in the form ofaddition salts with alkali or alkaline-earth metal or organic amines.

[0015] When in the compounds of formula (I) the bond between 3 and 4positions of benzopyran ring is a single bond, the two carbon atoms in3- and 4-positions of the ring can be asymmetric, and the compounds canexist as pure enantiomer, mixture of diastereoisomers or racemates.

[0016] Alkyl is preferably a C₁-C₄ alkyl, e.g. methyl, ethyl, n-propyl,isopropyl, tert-butyl.

[0017] Haloalkyl is preferably C₁-C₄ alkyl substituted by one to fivefluorine or chlorine atoms, e.g. trifluoromethyl.

[0018] Alkenyl is preferably a C₂-C₄ alkenyl, e.g. ethenyl or allyl.Haloalkenyl is preferably a C₂-C₄ alkenyl, substituted by one to threehalogen atoms.

[0019] Aryl is preferably phenyl or naphthyl, optionally substituted byone to three substituents, which are the same or different, selectedfrom halogen atoms, alkyl, alkoxy, nitro, cyano, trifluoromethyl, amino,hydroxy groups.

[0020] Alkanoyl is preferably a C₁-C₄ alkanoyl group, e.g. formyl,acetyl or propionyl group.

[0021] Aryloyl is preferably benzoyl optionally substituted by one tothree substituents, which are the same or different, selected fromhalogen atoms, alkyl, alkoxy, nitro, cyano, trifluoromethyl, amino,hydroxy groups.

[0022] Preferred compounds of formula (I) are those wherein R₁ and R₂,taken together with the nitrogen atom, form a piperidino or apyrrolidino ring, Y is hydrogen or an alkanoyl group,

[0023] Z is hydrogen, hydroxy or alkoxy, n is zero or 1, p is 2, X andX₁ are hydrogen or one is hydrogen and the other is halogen or an OR₃group wherein R₃ is as defined above, preferably in the para position.

[0024] In a preferred embodiment the invention relates to2H-1-benzopyran derivatives of formula (IA):

[0025] wherein X is H, C₁-C₄ alkoxy and Y is H, C₁-C₄ alkyl, C₁-C₅alkanoyl, aryloyl.

[0026] Aryloyl is preferably benzoyl optionally substituted by one tothree substituents, which are the same or different, selected fromhalogen atoms, alkyl, alkoxy, nitro, cyano, trifluoromethyl, amino,hydroxy groups.

[0027] The compounds can exist in the form of organic or inorganic acidaddition salts, or of addition salts with alkaline or alkaline-earthmetal or with organic amines.

[0028] The compounds of formula (I) belong to the class of the SelectiveEstrogen Receptor Modulators (SERMs) which bind and interact with theestrogen receptor but which act in certain tissues, such as bone, asestrogen agonists and in other tissues, and in particular in the breastand uterus, as estrogen antagonists.

[0029] The SERMs retain the beneficial effects of estrogen without someof its side effects.

STATE OF THE ART

[0030] WO 94/20098 in the name of Zymogenetics discloses3,4-diaryl-2H-1-benzopyrans for use in reducing bone loss and inparticular bone loss associated with osteoporosis.

[0031] A particularly preferred compound for use within the Zymogeneticsinvention is the 1-enantiomer of centchroman, later developed by NovoNordisk as levormeloxifene for the treatment of osteoporosis.

[0032] Levormeloxifene is hereinafter used as reference compound.

[0033] Substituted benzopyran and thiobenzopyran derivatives having apotential anti-estrogenic activity have been later on disclosed in WO98/25916 and WO 99/65893, in the name of C&C Research Laboratories.

[0034] The compounds of the invention differ in their chemical structurefrom the benzopyrans of the prior art for:

[0035] the presence of a double bond between the atoms in position 3-4or 4-4a, alternatively endo- or exo-cyclic;

[0036] the substitution on the carbon atom in 4 of the benzopyran ringwith a t-aminoalkyloxybenzyl/or benzylidenyl radical;

[0037] the presence of an hydroxy group in 5 when the substituent on thecarbon in 4 is a t-aminoalkyloxyphenyl radical.

[0038] These differences in the benzopyran structure and in particularthe presence of a methylene or methylidene bridge between the carbonatom in position 4 of the benzopyran ring and the carbon atom of thephenyl group has revealed of fundamental importance for thepharmacological activity of the compounds of the invention.

[0039] A non-steroidal benzothiophene derivative raloxifene, describedin Jones CD et al J Med Chem 27, 1057-159, 1984, provided withtissue-specific estrogen agonist and antagonist actions has beenadmitted for clinical use in the US and some European countries.

[0040] The compounds of the invention favourably compare also withraloxifene.

[0041] Other synthetic compounds with this possible spectrum ofactivities, including triphenylethylene and dihydronaphthalene have beendescribed as SERMs. However, the development of many of these compoundsas drugs has demonstrated to be problematic due to their excessivestimulation of uterine tissue.

[0042] The novel SERMs of the invention demonstrate significantbeneficial effects on bone and serum lipid levels and antagonisticeffects associated with a low degree of intrinsic estrogenicity onreproductive tissue.

[0043] For their tissue-specific estrogen-agonistic and antagonisticproperties the compounds of the invention can be used in therapy inparticular for the prevention and treatment of a number ofpostmenopausal pathologies, in particular osteoporosis, coronary heartdisease and estrogen dependent human cancer.

[0044] The present invention also provides a process for the preparationof a compound of formula (I)

[0045] wherein

[0046] R₁ and R₂ are independently H, alkyl, haloalkyl, alkenyl orhaloalkenyl, or together with nitrogen atom they are bound to can form a4- to 8-membered heterocyclic ring

[0047] X is H, alkyl, aryl, nitro, halo, O—R₃ wherein R₃ is H, alkyl,aryl, alkanoyl, aryloyl

[0048] X₁ is H, alkyl or alkoxy and when X and X₁ are alkyl they canform together with the carbon atom they are bound to, a fused aromaticring to give an α-naphthalenyl

[0049] Y is H, alkyl, alkanoyl, aryloyl

[0050] Z is H, O—R₄ wherein R₄ is H, alkyl, alkanoyl, aryloyl

[0051] m is 1,2

[0052] n is 0,1

[0053] p is 2-6

[0054]

represents a single or double bond between the atoms in positions 3-4 or4-4a: when n is 1 the double bond may be alternatively exocyclic orendocyclic to give respectively a 4-benzyliden chroman for m=1 or a4-benzyl-chrom-3-ene for m=2; when n is 0 the endocyclic bond 3-4 may bea single or a double bond and when n is 0 and the endocyclic bond 3-4 isa single bond, Z is not H.

[0055] In the schemes (1) and (2) are represented processes for thepreparation of 2-(N-piperidinyl) ethoxy compounds of formula (I). Thesereaction schemes can also be applied to other t-aminoalkyloxy analogousof formula (I).

[0056] Compounds of formula (I) wherein Z=OR₄ may be obtained accordingto scheme (1), by reacting 1,3,5-trihydroxybenzene with a substitutedphenylacetonitrile in an aprotic solvent saturated with gaseous HCl, toobtain the ketimines of formula (II) that can be hydrolyzed in boilingwater to give the corresponding ketones of formula (III) that can becyclized by means of N,N-dimethylformamide dimethylacetal and borontrifluoride diethyl etherate in dimethylformamide (DMF) to givecompounds of formula (IV).

[0057] Compounds of formula (IV) can be protected in the 7-position bymeans, for example, of an appropriate anhydride, with or without asolvent, at temperature from 0° C. to the boiling point of the solventor of the pure anhydride, or by means of an appropriate halide, in anaprotic solvent and in the presence of a base, to obtain an ester offormula (V) that has to be stable when submitted to the Grignardreaction.

[0058] The resulting compounds of formula (V) can be hydrogenated, inthe presence of a suitable catalyst, to give compounds of formula (VI).

[0059] The addition of Grignard reagents of formula (VII), obtained from4-(t-aminoalkyloxy)-1-bromobenzene and Mg in an aprotic solvent, to theketones of formula (VI), at temperature from −70° C. to the boilingpoint of the solvent, leads to the alcohol (VIII) that can bedehydrated, for example, in an ethanolic solution of conc. HCl, toobtain compounds of formula (IX).

[0060] Compounds of formula (IX) can be alkylated or acylated by meansof an alkyl or acyl halide in an appropriate solvent and in the presenceof a base, or can be acylated by means of an acyl anhydride at hightemperature and without a solvent, to give compounds of formula (X)where Y (different from R₄) is the protecting group selected from thosethat are stable to the Grignard reaction. Compounds of formula (IX) canbe deprotected, for example with conc. HCl or conc. HBr at hightemperatures, or, when Y=acyl, with K₂CO₃ in aqueous methanol at roomtemperature, to give the corresponding free phenols of formula (XI) thatcan be dialkylated, for example by means of an alkyl halide in anappropriate solvent and in the presence of a base, or can be diacylated,for example by means of an acyl halide in an appropriate solvent and inthe presence of a base or by means of an acyl anhydride, with or withouta solvent, to give compounds of formula (X) where Y is R₄ but differentfrom H; compounds of formula (X) can be hydrogenated in a suitablesolvent and in the presence of a catalyst, to give the correspondingcompounds of formula (XII) where Y is R₄ or different from R₄ and whereY can be different from that selected as protecting group in theGrignard reaction.

[0061] Compounds of formula (IX) can also be hydrogenated to givecompounds of formula (XIII).

[0062] Compounds of formula (I) wherein Z is H can be obtained followingscheme (2).

[0063] Compound (XIV) was protected at the phenolic hydroxy group withan appropriate protective moiety (Y) selected from those described inthe literature for the protection of phenols (see for examples T. W.Greene, P. G. Wuts “Protective groups in organic synthesis”, 3rd Ed,John Wiley & Sons, Inc., 1999, pages 246-292) to obtain compound (XV).The protective group must be selected from those that are stable in theconditions of the next steps. The compound (XV) was subsequentlyhydrogenated selectively at the olefinic double bond, in a manner toavoid the reduction of ketone to alcohols, by means of a catalytichydrogenation with an appropriate catalyst (containing Pd, Pt, Rh, Ru orother transition metals) in an appropriate solvent (alcohols, THF,acetone, dioxane, ethyl acetate, alcohols-water mixtures etc.) or by achemical reduction with an hydride in an appropriate aprotic solvent, togive compound (XVI). Compound (XVI) was reacted with compound (XVII)(obtained by reaction of protected 4-hydroxybenzyl chloride with Mg in asuitable solvent like diethyl ether, THF or other ethereal solvents attemperature between r.t. and the boiling temperature of the solvent), toobtain a compound of formula (XVIII). The protective group (Y′) for4-hydroxybenzyl chloride must be selected from the same as previouslydescribed, but generally different from Y selected for compound (XV).Selective removal of this second protective group (Y′) gave compound(XIX). Compounds of formula (XIX) can be alkylated with a haloalkylaminein a suitable solvent and in the presence of a base, to give compoundsof formula (XX).

[0064] Compounds of formula (XX) can be dehydrated (for example withaqueous HCl in acetonitrile at room temperature) to give a mixture ofcompounds of formulae (XXI) and (XXII) or can be deprotected (whenY=acyl) under mild conditions, for example with K₂CO₃ in aqueousmethanol at room temperature, to give compounds of formula (XXIII).(XXI) can be separated from the mixture by crystallization, while (XXII)can be purified by chromatographic methods.

[0065] Pure enantiomers of compounds of formula (XXI) can be obtained bychiral chromatography to give compounds of formula (XXV) and (XXVI).

[0066] Compounds of formula (XXI) and (XXII) can be hydrogenated to givecompounds of formula (XXVII). Compounds of formula (XXIII) can bedehydrated, for example, with aqueous HCl in acetonitrile, to givecompounds of formula (XXIV) that can be acylated or alkylated, forexample, with acyl or alkyl chloride in a suitable solvent and in thepresence of a base to give compounds of formula (XXII) in which Y can bedifferent from the one selected as protecting group in the Grignardreaction.

[0067] With an appropriate choice of Y it is possible to obtain compound(XXIV) directly from compound (XX) by acidic removal of Y protectivegroup and, simultaneously, hydrolysis of the tertiary alcohol.

[0068] Compounds of formula (XXI) where Y=acyl can be hydrolyzed forexample with K₂CO₃ in aqueous methanol to give the corresponding phenolsof formula (XXVIII) and these can be alkylated or acylated by means ofan alkylating or acylating agent, to obtain compounds of formula (XXI)in which Y can be different from the one selected as protecting group inthe Grignard reaction.

[0069] The preferred protecting group is pivaloyl(2,2-dimethylpropanoyl).

[0070] The following examples further illustrate the invention.

EXAMPLE 1 Preparation of 2,4,6-trihydroxyphenyl benzyl ketiminehydrochloride

[0071]

[0072] A solution of phloroglucinol (60 g) in diethyl ether (400 ml),refrigerated at 5° C., was added to a solution of phenylacetonitrile(50.1 g) and boron trifluoride diethyl etherate (4.7 ml) in HCl/ETOAc≅5M(800 ml), prepared by bubbling HCl gas in cooled ethyl acetate.

[0073] The solution was stored at 5° C. for 3 days. The crystallineyellow precipitate was filtered, washed with diethyl ether (50 ml) andused without further purification.

[0074] 95 g of product were obtained (Yield=80%)

[0075] TLC: chloroform/methanol=90/10 Rf=0.7

EXAMPLE 2 Preparation of 2,4,6 trihydroxyphenyl benzylketone

[0076]

[0077] 2,4,6-Trihydroxyphenyl benzylketimine hydrochloride (95 g) wassuspended in water (≅2000 ml) and warmed at 90° C. under stirring for 1hour.

[0078] After cooling, a crystalline product was obtained. The solid wasfiltered, washed with water (200 ml) and dried under vacuum at 60° C.

[0079] 70 g of product were obtained (yield 85%)

[0080] TLC: chloroform/methanol=90/10 Rf=0.8

EXAMPLE 3 Preparation of 5,7-dihydroxyisoflavone

[0081]

[0082] A solution of boron trifluoride diethyl etherate (228 ml) andN,N-dimethylformamide dimethylacetal (84 g) in N,N-dimethylformamide(715 ml) was added to a solution of the compound of example 2 (55 g) inN,N-dimethylformamide (1400 ml), warmed at 50° C. The mixture was heatedat 95° C. for 60 minutes.

[0083] The dark orange solution was cooled at r.t., poured into coldwater (10 l) and left overnight without stirring.

[0084] The pink solid was filtered, washed with water (1 l) and driedunder vacuum at 70° C.

[0085] 40.3 g of product were obtained (yield 70.4%)

[0086] TLC : hexane/ethyl acetate=70/30 Rf=0.55

EXAMPLE 4 Preparation of 7-pivaloyloxy-5-hydroxyisoflavone

[0087]

[0088] The compound of example 3 (10 g) and pivalic anhydride (50 ml)were heated at 120° C., under magnetic stirring, in a closed flask for 4h. The red dense solution was kept at r.t. without stirring overnight.

[0089] The precipitate was filtered, washed with petroleum ether anddried u.v. at 45° C.

[0090] 8.9 g of a pink solid were obtained. (yield 65%)

[0091] TLC : methylene chloride/methanol=95/5 R.f.=0.8

EXAMPLE 5 Preparation of 3-phenyl-5-hydroxy-7-pivaloyloxychroman-4-one

[0092]

[0093] A solution of the compound of example 4 (8.7 g) in acetone (300ml) was added with 5% Pd/C (9 g) (water content≅50%) and hydrogenated at40 psi in a Parr apparatus for 2.5 h. The catalyst was filtered on acelite pad and the filtrate evaporated to dryness. The oily residue wasdissolved in diethyl ether, dried (Na₂SO₄) and evaporated again. 8 g oforange oil were obtained (yield 90%).

[0094] It was used without further purification.

[0095] TLC: petroleum ether/EtOAc=95/5 R.f.=0.5

EXAMPLE 6 Preparation of 4-(2-(N-piperidinyl)ethoxy)-1-bromobenzene

[0096]

[0097] A solution of bromophenol (10 g) in dimethylformamide (160 ml)was added with K₂CO₃ (20.7 g) and heated at 100° C., under stirring, for10 min. N-(2-chloroethyl)piperidine hydrochloride (9.6 g) was added overa period of 10 min. The mixture was heated and stirred for 2 h, thencooled at r.t., poured into water (300 ml) and extracted with ethylacetate (300 ml). The organic phase was extracted with 3% HCl (2×200ml). After alcalinization, the aqueous phase was extracted with ethylacetate (300 ml); evaporation of the solvent gave 13 g of product as anorange oil.

[0098] TLC: methylene chloride/methanol/acetic acid=70/20/10 R.f.=0.7

EXAMPLE 7 Preparation of 4-(2-(N-piperidinyl)ethoxy)phenylmagnesiumbromide

[0099]

[0100] Magnesium (2.1 g) was charged into a 500 ml four necked flask,equipped with a condenser, a dropping funnel and a magnetic stirrer,under N₂ atmosphere.

[0101] 5 ml of a solution of the compound of example 6 (11.8 g) infreshly distilled tetrahydrofuran (THF) (50 ml) was added, then themixture was heated to reflux.

[0102] The remaining solution was dropped into the boiling reactionmixture over a period of 40 min. The mixture was allowed to reach r.t.,then the turbid gray solution was used in the following step.

EXAMPLE 8 Preparation of3-Phenyl-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-4,5-dihydroxy-7-pivaloyloxychromane hydrochloride

[0103]

[0104] The Grignard solution obtained from the previous step was quicklyadded to a solution of the compound of example 5 (2.6 g ) in dry THF (20ml), under stirring. Temperature raised to 40° C. After stirring for 1.5h, water (10 ml) was added and the mixture evaporated to dryness u.v.The crude product was dissolved in ethyl acetate (300 ml) and theinsoluble material filtered off. The organic solution was washed withHCl 0.25N (200 ml) (the hydrochloride salt of the product is moresoluble in ethyl acetate than in water), and concentrated to a littlevolume until precipitation occurred. After ultrasonication, the solidwas filtered and air dried.

[0105] 1.6 g of product were obtained as a white powder. (Yield 36%).

[0106] TLC: methylene chloride/methanol=90/10 R.f.=0.3

[0107] M.P.: 220-221° C.

[0108] NMR: complies.

EXAMPLE 9 Preparation of3-Phenyl-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-5-hydroxy-7-pivaloyloxychrom-3-ene hydrochloride

[0109]

[0110] The compound of example 8 (0.71 g) was dissolved in boiling ethylalcohol (40 ml). The solution was cooled at r.t. and HCl 37% (0.2 ml)was added. After 2 h at r.t, the solution was evaporated to completedryness and the solid foam was crystallized from acetone. The whitesolid obtained was filtered, washed with acetone and dried u.v. at 35°C.

[0111] 550 mg were obtained (yield 80%).

[0112] M.P.: 229-230° C.

[0113] T.L.C.: methylene chloride/methanol/triethylamine=95/5/1 R.f.=0.3

[0114] NMR: complies.

EXAMPLE 10 Preparation of3-Phenyl-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-5-hydroxy-7-pivaloyloxychromanehydrochloride

[0115]

[0116] 470 mg of the compound of example 9 were suspended in acetone (50ml); H₂O (2 ml) were added and the limpid solution was hydrogenated in aParr apparatus at 40 psi with 5% Pd/C (2 g, 50% H₂O) for 3 h.

[0117] After filtration of the catalyst the solution was evaporated andthe white solid foam obtained was dissolved in a little amount of MeOHand precipitaded by adding Et₂O.

[0118] 230 mg of compound were obtained.

[0119] T.L.C.: methylene chloride/methanol/triethylamine=90/10/0.5

[0120] M.P.: 260-270° C. (Dec.)

[0121] NMR: complies

EXAMPLE 11 Preparation of3-Phenyl-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-5,7-dihydroxychrom-3-enehydrochloride

[0122]

[0123] 370 mg of the compound of example 9 were dissolved in hot THF (20ml), then 130 mg of LiAlH₄ were added and the mixture was stirred for 30min.

[0124] Water (170 mg) was cautiously added, the gray solid was filteredoff and the solution acidified with HCl/EtOAc. The mixture wasevaporated, the residue dissolved in EtOH (2 ml) and ultrasonicated.

[0125] A white solid precipitated. It was filtered, washed with Et₂O anddried at 50° C. u.v.

[0126] 210 mg of compound were obtained.

[0127] M.P. 248-251° C. (Dec.)

[0128] T.L.C.: methylene chloride/methanol/triethylamine=95/5/1R.f.=0.25

[0129] NMR: complies

EXAMPLE 12 Preparation of 4′,7-dipivaloyloxy-5-hydroxyisoflavone

[0130]

[0131] Genisteine (10 g) and pivalic anhydride (75 ml), commerciallyavailable or prepared with known methods, were heated at 110° C. for 22h. then the brown solution was cooled and left at r.t. overnight.

[0132] A little amount of solid was formed; light petroleum ether (150ml) was added to the mixture then the abundant solid was filtered,washed with light petroleum ether and dried u.v. at 45° C.

[0133] 11.6 g of the title compound were obtained.

[0134] T.L.C.: light petroleum ether/EtOAc=85/15 R.f.=0.5.

EXAMPLE 13 Preparation of3-(4-pivaloyloxyphenyl)-5-hydroxy-7-pivaloyloxy chroman-4-one

[0135]

[0136] The compound of example 12 (10 g) was dissolved in acetone (750ml) and hydrogenated at 40 psi using 5% Pd/C (10 g; 50% H₂O content) ascatalyst.

[0137] After 3 h the catalyst was filtered off and the solution wasevaporated to obtain an oil that was crystallized from light petroleumether (150 ml).

[0138] 8.7 g of the title compound were obtained.

[0139] T.L.C.: light petroleum ether/EtOAc=85/15 R.f.=0.5

EXAMPLE 14 Preparation of3-(4-pivaloyloxyphenyl)-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-4,5-dihydroxy-7-pivaloyloxychromanehydrocloride

[0140]

[0141] The title compound was obtained with the same procedure describedin example 8 in which the compound of example 13 is used in place of thecompound of example 5.

[0142] T.L.C.: chloroform/methanol/30% NH₄OH=95/5/0.1 R.f.=0.35

EXAMPLE 15 Preparation of3-(4-pivaloyloxyphenyl)-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-5-hydroxy-7-pivaloyloxychrom-3-enehydrochloride

[0143]

[0144] The title compound was obtained with the same procedure describedin example 9 in which3-(4-pivaloyloxyphenyl)-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-4,5-dihydroxy-7-pivaloyloxychromane hydrochloride is used in place of the compound of example 8.

[0145] T.L.C.: chloroform/methanol/30% NH₂OH=95/5/0.1 R.f.=0.4

[0146] M.P.:243-245° C. (Dec.)

EXAMPLE 16 Preparation of3-(4-pivaloyloxyphenyl)-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-5-hydroxy-7-pivaloyloxychromanehydrochloride

[0147]

[0148] The title compound was obtained with the same procedure describedin example 10 in which the compound of example 15 is used in place ofthe compound of example 9.

[0149] T.L.C.: chloroform/methanol/30% NH₄OH=95/5/0.1 R.f.=0.45

[0150] M.P.: 205-207° C. (dec.)

EXAMPLE 17 Preparation of3-(4-pivaloyloxyphenyl)-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-5-isopropoxy-7-pivaloyloxychrom-3-enehydrochloride

[0151]

[0152] The compound of example 15 (210 mg ) was dissolved in DMF (4 ml),than K₂CO₃ (300 mg) and 2-iodopropane (200 mg) were added and themixture was stirred at r.t. for 2 h.

[0153] H₂O (5 ml) was added, the precipitated product was filtered,washed with H₂O and purified by flash chromatography over silica gel(eluent: methylene chloride/methanol=95/5 R.f.=0.7).

[0154] The product obtained was dissolved in acetone (2 ml) and addedwith a little excess of HCl in EtOAc to obtain the crystallinehydrochloric salt.

[0155] 7 mg of the title compound were obtained.

[0156] M.P.: 237-239° C.

[0157] NMR: complies

EXAMPLE 18 Preparation of 4′-methoxy-7-pivaloyloxy-5-hydroxyisoflavone

[0158]

[0159] Biochanin A (20 g), commercially available or prepared with knownmethods, was suspended in pivalic anhydride (100 ml) and the mixture washeated at 120° C., under stirring, for 4 h.

[0160] The brown solution was left at r.t. overnight; the precipitatedproduct was filtered, washed with light petroleum ether and dried at 70°C. u.v.

[0161] 19.6 g of the title compound were obtained.

[0162] T.L.C.: methylene chloride/methanol=95/5 R.f.=0.8

EXAMPLE 19 Preparation of3-(4-methoxyphenyl)-5-hydroxy-7-pivaloyloxychroman-4-one

[0163]

[0164] The title compound was obtained with the same procedure describedin example 5 in which 4′-methoxy-7-pivaloyloxy-5-hydroxyisoflavone isused in place of the compound of example 4.

[0165] TLC: petroleum ether/EtOAc=95/5 R.f.=0.5

EXAMPLE 20 Preparation of3-(4-methoxyphenyl)-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-4,5-dihydroxy-7-pivaloyloxychromanehydrochloride

[0166]

[0167] The title compound was obtained with the same procedure describedin example 8 in which3-(4-methoxyphenyl)-5-hydroxy-7-pivaloyloxychroman-4-one is used inplace of the compound of example 5.

[0168] T.L.C.: chloroform/methanol/30% NH₄OH=95/5/0.1 R.f.=0.3

EXAMPLE 21 Preparation of3-(4-methoxyphenyl)-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-5-hydroxy-7-pivaloyloxychrom-3-enehydrochloride

[0169]

[0170] The title compound was obtained with the same procedure describedin example 9 in which3-(4-methoxyphenyl)-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-4,5-dihydroxy-7-pivaloyloxychromanehydrocloride is used in place of the compound of example 8.

[0171] T.L.C.: chloroform/methanol/30% NH₄OH=95/5/0.1 R.f.=0.35

[0172] M.P.: 214-216° C. (dec.)

[0173] NMR: complies

EXAMPLE 22 Preparation of3-(4-methoxyphenyl)-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-5,7-dihydroxychrom-3-ene hydrochloride

[0174]

[0175] The title compound was obtained with the same procedure describedin example 11 in which the compound of example 21 is used in place ofthe compound of example 9.

[0176] T.L.C.: chloroform/methanol/30% NH₄OH=95/5/1 R.f.=0.3

[0177] M.P.: 198-200° C. (dec.)

EXAMPLE 23 Preparation of 7-pivaloyloxyisoflavone

[0178]

[0179] To a solution of 7 g of 7-hydroxyisoflavone in 190 ml ofN,N-dimethylformamide, 2 g of sodium hydride (80% in paraffin oil) wereadded. The mixture was stirred for 10 minutes, then 4.2 g of pivaloylchloride were dropped in 1 minute, under vigorous stirring. After 20minutes at r.t. the mixture was poured into water (400 ml), theprecipitate was filtered, washed with water (1000 ml) and dissolved inchloroform (800 ml). The organic solution was dried over sodium sulphateand evaporated to dryness.

[0180] 7.6 g of white solid were obtained.

[0181] TLC eluent: light petroleum ether/ethyl acetate=85/15 R.f.=0.6

EXAMPLE 24 Preparation of 3-phenyl-7-pivaloyloxychroman-4-on

[0182]

[0183] A solution of 15.7 of the compound of example 23 in 770 ml of1,4-dioxane was added with 8 g of wet 5% palladium on carbon andhydrogenated in a Parr apparatus at 40 psi. The reaction was monitoredby T.L.C. to avoid further reduction to 4-hydroxy derivatives. After 2hours the catalyst was filtered on a celite pad and washed with1,4-dioxane (200 ml).

[0184] The solution was evaporated to dryness and the residue oil (21.35g) was crystallized by dissolving it in 115 ml of ethyl acetate, adding635 ml of light petroleum ether and storing at −20° C. overnight.

[0185] 8.53 g of product were obtained.

[0186] Crystallization of mother liquor in ethyl alchool gave a secondcrop of 1.3 g of pure product.

[0187] TLC: light petroleum ether/ethyl acetate=80/20 Rf=0.85

EXAMPLE 25 Preparation of3-phenyl-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-4-hydroxy-7-pivaloyloxychromanehydrochloride

[0188]

[0189] The title compound was obtained with the same procedure describedin example 8 in which the compound of example 24 is used in place ofexample 5.

[0190] T.L.C.: methylene chloride/methanol=90/10 R.f.=0.4

EXAMPLE 26 Preparation of3-phenyl-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-4-hydroxy-7-pivaloyloxychrom-3-enehydrochloride

[0191]

[0192] The title compound was obtained with the same procedure describedin example 9 in which the compound of example 25 is used in place of thecompound of example 8.

[0193] T.L.C.: methylene chloride/methanol/30% NH₄OH=95/5/1 R.f.=0.4

[0194] M.P.: 233-235° C. (dec.)

EXAMPLE 27

[0195] Preparation of 3-phenyl-5-hydroxy-7-methoxychroman-4-one

[0196] The title compound was obtained with the same procedure describedin example 5 in which 5-hydroxy-7-methoxyisoflavone is used in place ofthe compound of example 4.

EXAMPLE 28 Preparation of3-phenyl-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-4,5-dihydroxy-7-methoxychromanehydrochloride

[0197]

[0198] The title compound was obtained with the same procedure describedin example 8 in which 3-phenyl-5-hydroxy-7-methoxychroman-4-one is usedin place of the compound of example 5.

[0199] T.L.C.: methylene chloride/methanol=90/10 R.f.=0.25

EXAMPLE 29 Preparation of3-phenyl-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-5-hydroxy-7-methoxychrom-3-enehydrochloride

[0200]

[0201] The title compound was obtained with the same procedure describedin example 9 in which the compound of example 28 is used in place of thecompound of example 8.

[0202] T.L.C.: methylene chloride/methanol/30% NH₄OH=95/5/1 R.f.=0.3

[0203] M.P.: 253-256° C. (dec.)

[0204] NMR: complies

EXAMPLE 30 Preparation of3-Phenyl-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-5-methoxy-7-pivaloyloxychrom-3-enehydrochloride

[0205]

[0206] The compound of example 9 (550 mg) was dissolved in DMF (30 ml),then a solution of K₂CO₃ (400 mg) in H₂O (3.5 ml) and a solution of CH₃I(0.9 ml) in DMF (9.5 ml) were added, and the mixture was stirred for 5h, quenched in H₂O (200 ml) and extracted with EtOAc (200 ml). Theorganic phase was washed with brine (2×200 ml), dried over sodiumsulfate and evaporated u.v. The yellow oil was purified byflash-chromatography over silica gel (eluent: CH₂Cl₂/MeOH/NEt₃=95/5/0.1R.f.=0.4). The product obtained was transformed into its hydrochloridesalt by adding HCl/EtOAc to a solution of the free base in EtOAc. 190 mgof white amorphous solid was obtained.

[0207] T.L.C.: methylene chloride/methanol/NEt3=95/5/0.1 R.f.=0.4

[0208] NMR: complies

EXAMPLE 31 Preparation of3-(4-fluorophenyl)-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-5-hydroxy-7-pivaloyloxychrom-3-enehydrochloride

[0209]

[0210] The title compound was obtained following the proceduresdescribed in examples 1 to 9 in which the starting material was4-fluorophenylacetonitrile in place of phenylacetonitrile of example 1.

[0211] T.L.C.: methylene chloride/methanol/30% NH₄OH=95/5/1 R.f.=0.3

[0212] M.P.: 198-200° C. (dec.)

[0213] NMR: complies

EXAMPLE 32 Preparation ofcis-3-phenyl-4-(4-benzyloxybenzyl)-4-hydroxy-7-pivaloyloxychromane

[0214]

[0215] Magnesium (4.9 g) was charged, under N₂, in a 500 ml four neckedflask, equipped with a condenser, a dropping funnel and a magneticstirrer.

[0216] 4-benzyloxybenzyl chloride (14.7 g) was dissolved in 190 ml offreshly distilled THF and charged into the dropping funnel.

[0217] 10 ml of this solution were dropped over the magnesium at r.t.,and the mixture strongly heated till effervescence is evident on themagnesium surface. The flask was immediately dipped into an oil bath at75° C. and the remaining solution was added over a period of 40 min. Themixture was allowed to reach r.t., then the gray turbid solution wastransferred into a 500 ml flask, equipped with a thermometer, a CaCl₂valve, a magnetic stirrer and a dropping funnel, under N₂. The Grignardsolution was immediately used in the next step or stocked in freezer.(Yield 100%, calculated weighing the unreacted magnesium).

[0218] A solution of the compound of example 24 (10 g), in freshlydistilled THF(100 ml), was dropped into the above solution, refrigeratedat ≅−20° C., over a period of 30 min.

[0219] The yellow-orange mixture was allowed to reach r.t., stirred for2 hours, cooled at 0° C. and added with water (8 ml), under stirring.The solid formed was discharged, the solution was evaporated to drynessand triturated with EtOAc/light petroleum ether=15/85 (2×300 ml).

[0220] After filtration of the solid (Mg salts and other byproducts),the solution was evaporated and the oily residue was crystallized fromEtOH (200 ml).

[0221] 7.0 g of white solid was obtained; a second crop (1.4 g) of purecis-3-phenyl-4-(4-benzyloxybenzyl)-4-hydroxy-7-pivaloyloxychromane wasobtained by flash chromatography (EtOAc/light petroleum ether=15/85) ofmother liquids.

[0222] T.L.C.: EtOAc/light petroleum ether=15/85 R.f.=0.35

EXAMPLE 33 Preparation ofcis-3-phenyl-4-(4-hydroxybenzyl)-4-hydroxy-7-pivaloyloxychromane

[0223]

[0224] The compound of example 32 (6.9 g) was dissolved in MeOH (800 ml)and hydrogenated in a Parr apparatus with 14 g of wet 5% Pd /C at 40 psifor 45 min.

[0225] After filtration of the catalyst on a celite pad, the solutionwas evaporated under reduced pressure, and the oily residue was usedwithout further purification in the following step.

[0226] T.L.C: EtOAc/light petroleum ether=30/70 R.f.=0.5

EXAMPLE 34 Preparation ofcis-3-phenyl-4-(4-(2-(N-piperidinyl)ethoxy)benzyl)-4-hydroxy-7-pivaloyloxychromane

[0227]

[0228] To a solution of the compound of example 33 (5.3 g) in acetone(170 ml), N-(2-chloroethyl)piperidine hydrochloride (2.48 g) and K₂CO₃(3.38 g) were added, than the mixture was refluxed under stirring for 16hours.

[0229] The solid was filtered off, the solution was evaporated and theoily residue was dissolved in boiling hexane (500 ml).

[0230] After filtration of the insoluble material, the hot solution wascooled at r.t. and ultrasonicated (or vigorously stirred) untilcrystallization occurred. After storage at −20° C., the solid wasfiltered and dried u.v. at 50° C.

[0231] 3.2 g of a white solid were obtained.

[0232] Mother liquids can be submitted to flash chromatography(CH₂Cl₂/MeOH/NEt₃=95/5/0.1) to give additional 1.2 g of product.

[0233] T.L.C.: methylene chloride/methanol/NEt₃=95/5/0.1 R.f.=0.4

EXAMPLE 35 Preparation ofrac-3-phenyl-4-E-(4-(2-(N-piperidinyl)ethoxy)benzyliden-7-pivaloyloxychromane hydrochloride

[0234]

[0235] A solution of the compound of example 34 (4.2 g) in CH₃CN (300ml), refrigerated at −20° C., was added to a cold (−20° C.) solution of37% HCl (3 ml) in CH₃CN (300 ml), and left at −20° C. overnight.

[0236] The solution was allowed to reach r.t. and checked by H.P.L.C.

[0237] When the compound of example 34 was completely consumed, thesolvent was evaporated under vacuum, the residue was dissolved inacetone and evaporated again, to obtain a white solid foam that wasdissolved in EtOAc and crystallized by ultrasonication or by stirring.

[0238] T.L.C.: EtOAc/n-ButOH/H₂O/CH₃COOH=50/10/10/10 R.f.=0.3

[0239] M.P.:214-216° C.

[0240] NMR: complies

EXAMPLE 36 Separation of Enantiomers of the Compound of Example 35

[0241] The compound of example 35 was submitted to preparative H.P.L.C.separation.

[0242] The conditions of the preparative separation are listed below:

[0243] Column:Chorisis 10 μm, 7.8×300 mm

[0244] Eluent: Acetonitrile/0.5% acetic acid=25/75

[0245] Flow: 2 ml/min

[0246] λ: 254 nm

[0247] Charge: 25 mg dissolved in 0.5 ml of mobil phase.

[0248] The enantiomeric purity of the two enantiomers was determined byanalytical chiral chromatography; conditions are listed above:

[0249] Column:Chiralcel OJ-R, 5 μm, 4.6×150 mm

[0250] Eluent: Acetonitrile/0.01M NaH₂PO₄+1 ml HCLO₄(pH=5.6)=65/35

[0251] Flow: 0.5 ml/min

[0252] λ: 254 nm

[0253] Retention time of the (+) isomer: 5.6 min

[0254] Retention time of the (−) isomer: 6.2 min

[0255] The two isomers were collected as free base

[0256] They were converted into their hydrochloride salt by bubbling HClgas into an ethereal solution of the free bases.

[0257] [α]_(D)=+44.6 (c=1, MeOH)

[0258] [α]_(D)=−44.0 (c=1, MeOH)

EXAMPLE 37 Preparation ofrac-3-phenyl-4-E-(4-(2-(N-piperidinyl)ethoxy)benzyliden-7-hydroxychromanehydrochloride

[0259]

[0260] A solution of K₂CO₃ (440 mg) in H₂O (2.5 ml) was added to asolution of the compound of example 35 (440 mg) in MeOH (50 ml) andstirred overnight. 1N HCl (6.3 ml) was added, then the solution waspartially evaporated until a white emulsion was formed. After cooling to0° C., a solid separated that was filtered, washed with water andair-dried. 320 mg of white amorphous solid was obtained.

[0261] T.L.C.: EtOAc/n-ButOH/H₂O/CH₃COOH=50/10/10/10 R.f.=0.2

[0262] NMR: complies

EXAMPLE 38 Preparation ofcis-3-phenyl-4-(4-(2-(N-piperidinyl)ethoxy)benzyl)-4,7 dihydroxychromane

[0263]

[0264] A mixture of the compound of example 34 (12.5 g), MeOH (800 ml),K₂CO₃ (12.5 g) and H₂O (40 ml) was stirred for 4 h, the solvent wasevaporated to 50 ml and H₂O (500 ml) was added, under magneticalstirring. A white solid separated and was collected by filtration,washed with water and dried at 45° C. 9.2 g of the title compound wereobtained.

[0265] T.L.C.: methylene chloride/methanol/30% NH₄OH=93/7/0.7 R.f.=0.35

[0266] M.P. 95-103° C.

[0267] NMR: complies

EXAMPLE 39 Preparation of3-phenyl-4-(4-(2-(N-piperidinyl)ethoxy)benzyl)-7-hydroxychrom-3-ene

[0268]

[0269] A solution of 37% HCl (3 ml) in acetonitrile (40 ml) was added toa boiling solution of the compound of example 38 (9 g) in acetonitrile(300 ml). After 20 min the solution was quickly refrigerated to 0° C.,added with K₂CO₃ (9.0 g), stirred for 3 hours and the precipitated freebase together with K₂CO₃ was filtered, washed with water and dried undervacuum at 40 C.

[0270] To obtain 7 g of the title compound:

[0271] M.P.: 168-170° C. (Dec.)

[0272] T.L.C.: methylene chloride/methanol/30% NH₄OH=93/7/0.7 R.f.=0.4

[0273] NMR: complies

EXAMPLE 40 Preparation of3-phenyl-4-(4-(2-(N-piperidinyl)ethoxy)benzyl)-7-pivaloyloxychrom-3-enehydrochloride

[0274]

[0275] Pivaloyl chloride (0.5 g) was added to a mixture of the compoundof example 39 (0.5 g) and K₂CO₃ (0.5 g) in acetonitrile (40 ml); themixture was stirred for 5 h, the salts were filtered off, then HCl inEtOAc (slight excess) was added and the solution was evaporated todryness to leave a solid foam that was triturated with ethyl ether,filtered and dried al r.t.

[0276] 400 mg of the title compound were obtained as a white amorphoussolid.

[0277] T.L.C.: EtOAc/n-ButOH/H₂O/CH₃COOH=50/10/10/10 R.f.=0.4

[0278] NMR: complies

EXAMPLE 41 Preparation of3-phenyl-4-(4-(2-(N-piperidinyl)ethoxy)phenyl)-5,7-diacetyloxychrom-3-enehydrochloride

[0279]

[0280] The compound of example 11 (210 mg, prepared as in example 11)was dissolved in acetic anhydride (2 g) at 130° C. and stirred for 30min. The solution was cooled to room temperature, added with ice (2 g),diluted with water (50 ml) treated with aqueous K₂CO₃ and extracted withEtOAc (50 ml). The organic layer was dried over sodium sulfate,filtered, acidified with a slight excess of HCl/EtOAc and evaporated todryness. The yellow oil resulting was crystallized from acetone/Et₂O togive 100 mg of the title compound as a light-yellow powder.

[0281] T.L.C. methylene chloride/methanol/NEt₃=95/5/0.1 R.f.=0.55

[0282] M.P. 154-156° C. (Dec.)

[0283] NMR: complies

EXAMPLE 42 Preparation of3-phenyl-4-(4-(2-(N-piperidinyl)ethoxy)benzyl)-7-isobutyroyloxychrom-3-enehydrochloride

[0284]

[0285] The title compound was obtained following the proceduresdescribed in example 40 in which isobutyroyl chloride is used in placeof pivaloyl chloride.

[0286] T.L.C.: EtOAc/n-ButOH/H₂O/CH₃COOH=50/10/10/10 R.f.=0.4

[0287] M.P.=152-155° C.

[0288] NMR: complies

EXAMPLE 43 Preparation of3-phenyl-4-(4-(2-(N-piperidinyl)ethoxy)benzyl)-7-hydroxychrom-3-enemethansulfonate (1:1) (soluble salt of the compound of example 39)

[0289] 2.7 g of the compound of example 39 were dissolved in 120 ml ofhot acetone, then a solution of 512 mg of methansulphonic acid in 30 mlof acetone was added and the solution was magnetically stirred untilcrystallization occurred.

[0290] After standing at −20° C. for 2 h, the solid was filtered, washedwith Et2O and dried under vacuum at 40° C.

[0291] 2.9 g of a white solid were obtained. M.P.=170-175° C. (Dec.)

[0292] The solubility of this salt in water at r.t. was >1 mg/ml.

EXAMPLE 44 Preparation of3-phenyl-4-(4-(2-(N-piperidinyl)ethoxy)benzyl)-7-hydroxychrom-3-ene byisomerization of the compound of example 37

[0293] 25 ml of 1% HCl conc. in CH₃CN were added to a suspension of thecompound of the example 37 (660 mg) in CH₃CN/H₂O (150/5 ml) heated at80° C. The reaction was monitored in HPLC (column Select B RP C₁₈,eluent: CH₃CN—MeOH=80-20 70%, buffer 30%, flow=0.8 ml/min, λ=254 nm).After 15 min of heating at 80° C. the reaction finished: 85% of theproduct (retention time=4.8 min) formed and 11% of the reagent remainedunreacted. The reaction didn't proceed further, neither continuing theheating, nor adding a larger amount of acid.

[0294] The reaction mixture was worked up and the product was isolatedas described in the example 39.

[0295] 500 mg of the product were obtained (yield 75%).

EXAMPLE 45 Preparation of 2,2-dimethyl-propanoic acid3-(4-methoxyphenyl)-4-oxo-4H-1-benzopyran-7-yl ester

[0296]

[0297] Formononetine (90 g; 335 mmol) was suspended in acetonitrile,K₂CO₃ was added and the mixture was stirred for about 20 minutes at r.t.Pivaloyl chloride (59.85 g; 496 mmol) was dropped into the mixture, thenthe reaction was stirred for further 15 minutes and quenched into water.The solid was filtered, washed with water, dissolved in CHCl₃; thesolution was dried and evaporated to dryness to obtain the titlecompound, which can be crystallised from hot toluene. 102 g of a whitecrystalline powder were obtained.

[0298] M.P.: 159-161° C.; NMR, MS and IR comply with the structure.

EXAMPLE 46 Preparation of 2,2-dimethyl-propanoic acid3,4-dihydro-3-(4-methoxy phenyl)-4-oxo-2H-1-benzopyran-7-yl ester

[0299]

[0300] Compound of Ex 45 dissolved in dioxane and hydrogenated in a Parrapparatus at 35 psi with Pd/BaSO₄ 5% as catalyst.

[0301] After 3 hours the catalyst was filtered off, the solution wasevaporated to a little volume and quenched into water to obtain aprecipitate that was filtered and dissolved in hot 95% ethanol. Afterstanding overnight in a refrigerator, the white product was filtered andwashed with petroleum ether, to obtain 66 g of the title compound.

[0302] M.P.: 119-121° C.; NMR, MS and IR comply with the structure.

EXAMPLE 47 Preparation of 2,2-dimethyl-propanoic acid3,4-dihydro-4-hydroxy-3-(4-methoxyphenyl)-4-[[4-(phenylmethoxy)phenyl]methyl]-2H-1-benzopyran-7-ylester

[0303]

[0304] Mg turnings (15.4 g, 635 mmol) were loaded into a 2 liter flask;4-benzyloxybenzyl chloride (49.6 g, 211.6 mmol) was dissolved in dry THFand this solution was dropped in one hour on the Mg turnings under aninert atmosphere while maintaining a gentle reflux.

[0305] The mixture was refrigerated to −20° C., then a solution ofcompound of ex. 46 (60 g, 169.3 mmol) in dry THF (300 ml) was dropped ina 15 minutes period. After leaving the mixture to reach r.t., water (50ml) was added, the solid material was filtered off and the solution wasevaporated to dryness. Ethyl acetate (200 ml) was added to the residueoil and the mixture was allowed to stay at −20° C. for one hour. Thesolid was filtered off and the solution was evaporated to dryness. Theresidue oil was crystallised from a 80/20 mixture of 95% ethanol andpetroleum ether to give 60.8 g (110 mmol) of the title compound.

[0306] M.P.: 104-111° C.; NMR, MS and IR comply with the structure.

EXAMPLE 48 Preparation of 2,2-dimethyl-propanoic acid3,4-dihydro-4-hydroxy-4-[(4-hydroxyphenyl)methyl]-3-(4-methoxyphenyl)-2H-1-benzopyran-7-ylester

[0307]

[0308] Compound of ex. 47 (60.8 g, 110 mmol) was dissolved in ethylacetate, 5% Pd/C was added and the mixture was hydrogenated.

[0309] The catalyst was filtered off and the solution was evaporated todryness obtaining a solid foam (50.88 g , 110 mmol) that was usedwithout any further purification.

[0310] NMR, MS and IR comply with the structure.

EXAMPLE 49 Preparation of 2,2-dimethyl-propanoic acid3,4-dihydro-4-hydroxy-3-(4-methoxyphenyl)-4-[[4-[2-(1-piperidinyl)ethoxyl]phenyl]methyl]-2H-1-benzopyran-7-yleste

[0311]

[0312] A mixture of: compound of ex. 48 (50 g, 108.1 mmol),N-(2-chloroethyl)piperidine hydrochloride (20 g, 108.7 mmol) and K₂CO₃in boiling acetone (860 ml) was allowed to react.

[0313] The solid was filtered off and the solution was evaporated todryness obtaining an oil (62 g, 108.1 mmol) that was used without anyfurther purification.

[0314] NMR, MS and IR comply with the structure.

EXAMPLE 50 Preparation of3,4-dihydro-4-hydroxy-3-(4-methoxyphenyl)-4-[[4-[2-(1-piperidinyl)ethoxyl]phenyl]methyl]-2H-1-benzopyran-4,7-diol

[0315]

[0316] To a solution of compound of ex. 49 (60 g, 104.6 mmol) inmethanol, K₂CO₃ dissolved in water was added and allowed to react atr.t. for 3 hours. The solvent was evaporated to 300 ml and the mixturewas quenched into water and stirred overnight. The solid was filtered,washed with water and dried under vacuum at r.t. obtaining 36 g of crudeproduct that was crystallised from boiling acetonitrile to; obtain 28 gof the compound.

[0317] M.P.: 107.5-108.5° C.; NMR, MS and IR comply with the structure.

EXAMPLE 51 Preparation of3-(4-methoxy)phenyl-4-[[4-[2-(1-piperidinyl)ethoxyl]phenyl]methyl]-2H-1-benzopyran-7-olhydrochloride

[0318]

[0319] Compound of ex. 50 (24 g, 49 mmol) was dissolved in hotacetonitrile (550 ml), 37% HCl (4.9 ml) was added and the solution wasstirred at reflux for 45 minutes. The mixture was allowed to reach r.t.and the crystallised solid was filtered and washed with diethyl ether.17.3 g (34 mmol) of the title compound were obtained.

[0320] M.P.: 199-205° C. (dec.); NMR, MS and IR comply with thestructure.

[0321] The compounds of the invention have been tested for theirpharmacological activity.

[0322] In the following examples the results obtained with the compounds3-phenyl-4-(4-(2-(N-piperidinyl)ethoxy)benzyl)-7-hydroxycrom-3-ene (thecompound of example 39 employed as a free base or methansulphonic salt)and3-(4-methoxy)phenyl-4-[[4-[2-(1-piperidinyl)ethoxyl]phenyl]methyl]-2H-1-benzopyran-7-olhydrochloride (the compound of example 51), cited in the following asCompound I (example 39) and Compound II (example 51) are reported.

[0323] In Vitro Study—Human Estrogen Receptor-α and -β (ER-α and ER-β)

[0324] The experiment has been conducted as described in Obourn J D etal, Biochemistry 32, 6229, 1993.

[0325] The Compound I of the invention binds with high affinity to thehuman ER-α and ER-β Binding K_(i) were 0.041±0.011 and 0.157±0.028 nM,respectively.

[0326] Compared with the well-known SERM raloxifene, its ER-α affinitywas similar (K_(i)=0.071±0.008 nM), whereas the affinity for ER-β was 10fold higher (K_(i)=1.62±0.348 nM).

[0327] The Compound II of the invention binds with even higher affinityto the human estrogen receptor ER-α and ER-β. Binding K_(i) were0.017±0.002 and 0.099±0.005 nM, respectively.

[0328] Compared with the well known SERM raloxifene (K_(i)ER-β=1.62±0.348, ER-α=0.071±0.008 nM), the affinity for ERβ was 16 foldhigher, is whereas the affinity for ER-α was similar.

[0329] In vivo Studies—Immature Female Rat Assay.

[0330] Antiuterotrophic Activity

[0331] Experiments have been conducted as described in Eppenberger U etal, Am J Clin Oncol 14 (suppl. 2), S5-S14, 1991.

[0332] In immature female rats, treatment with 17α-ethynil estradiol(E2) at 0.05 mg/kg per os for 3 days significantly increased uterine wetweight (170-220%) compared with vehicle-treated controls.

[0333] This concentration of E2 was the lowest producing near-maximaleffect and was chosen on the basis of preliminary dose-responseexperiments.

[0334] The Compounds I and II administered orally inhibited theuterotrophic action of E2 in a dose related fashion. Total antagonismwas observed with Compound I at 1-10 mg/kg/day (EC₅₀=0.33 mg/kg·day),whereas the reference compound levormeloxifene only partiallyantagonized E2 stimulation of the uterus (40-45% inhibition at 1mg/kg/day).

[0335] Total antagonism was observed with Compound II at 0.1-1mg/kg·day. Comparison of the antioestrogenic potencies (ED50 =doserequired to produce 50% reduction in estradiol-stimulated growth) showedthat the Compound II of the invention was about 25 times more potentthan raloxifene (ED50=0.016 and 0.39 mg/kg·day, respectively).

[0336] Uterotrophic Activity

[0337] The Compound I and raloxifene at 0.1 mg/kg/day slightly increaseduterine weight compared with vehicle treated control rats (17 and 32%,respectively, significantly less than 0.1 mg/kg/day E2); however,uterine weight for 0.01, 1 and 10 mg/kg/day of the Compound I andraloxifene were not significantly different from control rats.

[0338] Uterine weights for 0.01, 0.1, 1 and 10 mg/kg.day of the CompoundII and raloxifene were not significantly different from control rats.

[0339] In line with previously reported data (M. Salman et al., J. Med.Chem. 1986, 29, 1801-1803), levormeloxifene at the same dose levelssignificantly increased uterine wet weight in a dose-depend way and amaximal agonist activity of 60-65% that of E2 was apparent at 1 mg/kg.

[0340] We can conclude that in the immature female rat assay, thepreferred compounds of the invention appeared potent and effective inantagonizing estrogen stimulation of the uterus down to the level ofvehicle treated controls with no significant estrogenicity.

[0341] The Compound II appeared more potent than raloxifene inantagonizing estrogen stimulation of the uterus.

[0342] In contrast, levormeloxifene was a partial agonist, demonstratingat 1 mg/kg-day a maximal agonist activity of 60-65% that of E2.

[0343] The pharmacological profile of the claimed compounds in immaturerats appears strictly different compared with the one of thestructurally related compound levormeloxifene, indicating that thestructural differences between the compounds of the invention and thoseof the prior art are relevant in reducing the uterine-stimulatingeffects.

[0344] Effects in the Ovariectomized (OVX) Rat Model

[0345] The experiments have been conducted according to Kaln D N, BoneMineral 15, 175-192, 1991 and Grese T A et al J Med Chem 40, 146-167,1997.

[0346] Four-Week OVX Rat Assay

[0347] The effects of the tested compounds were evaluated also in9-11-month-old OVX rats that were dosed for 4 weeks post-surgery andcompared with OVX and Sham controls.

[0348] Tissue-specific estrogen agonist effects were examined utilizinguterine weight, uterine histology, uterine eosinophil peroxidaseactivity (EPO), bone mineral density (BMD), and serum cholesterol levelsas end points.

[0349] The administration for 4 weeks was not associated with any overtsigns of toxicity.

[0350] Effect on Bone Mineral Density (BMD) (Lumbar Spine L1-4)

[0351] Bone mineral density (BMD) was measured by DEXA (Dual Energy XRayAbsorptiometry) using a Hologic QDR-1000 plus instrument equipped withdedicated software for small animal measurements. Anultrahigh-resolution mode (line spacing 0.0254 cm and resolution 0.0127cm) was used with a collimator of 0.63 mm diameter. This techniqueprovides an integrated measure of both cortical and trabecular bone.

[0352] In vivo DEXA measurements were carried out immediately beforesurgery (baseline scan) and 4 weeks after surgery. The anatomic regionexamined was the lumbar spine L1-4.

[0353] All animals were anesthetized before scanning with a mixture ofketamine and p-promazine. For each scan a rat was placed in a supineposition with the spine parallel to the long axis of the densitometertable. The lumbar spine was scanned using the pelvic bones as landmark;analysis of this site was accomplished by dividing vertebra andintervertebral spaces with subregional high resolution software andincluding only target vertebra in the global region of interest.

[0354] Percent protection was calculated by the following formula: %protection=[(% chance BMD_(test compound)−% chance BMD_(OVX control))/(%chance BMD_(sham control)−% chance BMD_(OVX control))]×100.

[0355] In a first experiment the bone sparing effects of Compound I weredetermined.

[0356] 4 weeks after surgery a significantly lower percent change in BMDfrom baseline in OVX rats compared to sham rats was observed (−9.39±0.60and −0.11±0.75%, respectively P<0.01).

[0357] As shown in the literature, 0.1 mg/kg of 17α-ethynil estradiol(E2) partially prevented the loss of bone (˜50% protection).

[0358] 0.1-, 1 and 10 mg/kg/day of the Compound I treatment reduced thebone loss, as their % change in BMD from the baseline was significantlyhigher compared to OVX rats (40,46 and 47% protection, respectively,P<0.01).

[0359] In a second experiment the bone sparing effects of Compound IIwere determined.

[0360] 4 weeks after surgery a significantly higher bone loss frombaseline in OVX rats compared to sham rats was observed (% change in BMD−5.16% and +1.28%, respectively P<0.01).

[0361] As shown in the literature, 0.1 mg/kg of 17α-ethynil estradiol(E2) prevented the loss of bone (˜95% protection).

[0362] 0.1 and 1 mg/kg·day of the tested compound treatment reduced thebone loss in a dose dependent manner, as their % change in BMD from thebaseline was significantly higher compared to OVX rats (60 and 119%protection, respectively, P<0.01).

[0363] The bone sparing effects of the tested compounds was comparablewith those achieved with E2 0.1 mg/kg·day, indicating that they are fulloestrogen agonists on bone.

[0364] Raloxifene and levormeloxifene were compared to Compoud II: theirprotective effect was significant (50-60% protection at 1 and 10mg/kg·day) but significantly lower compared to the compound of theinvention.

[0365] Effect on Serum Cholesterol Levels

[0366] 0.1-10 mg/kg day of the Compound I dose-dependently decreasedtotal serum cholesterol in OVX rats with half maximal efficacy ED₅₀ of0.12 mg/kg and maximal lowering of cholesterol observed at 10 mg/kg.(67% inhibition relative to the OVX control).

[0367] 0.1 and 1 mg/kg·day of the Compound II dose-dependently decreasedtotal serum cholesterol in OVX rats (75 and 81% inhibitionrespectively). Compound II was as efficacious as E2 in lowering serumcholesterol levels with respect to controls, whereas raloxifene andlevormeloxifene appeared significantly less potent.

[0368] Uterine Effects

[0369] In OVX rats treated with the compounds of the invention at 0.1, 1and 10 mg/kg·day, uterine weight was significantly lower than both shamcontrols and E2-treated OVX rats.

[0370] The degree of eosinophilic peroxidase induction of the uterus maybe a useful marker for estrogen-effected growth responses (Lyttle C Rand DeSombre E R Proc Natl Acad Sci USA 74, 3162-3166, 1977). The testedcompounds had no significant effect on eosinophilic peroxidase activitycompared to OVX uteri, whereas levormeloxifene significantly increasedthis parameter.

[0371] Moreover, the tested compounds administration was not associatedwith significant stimulation of uterine epithelia.

[0372] Instead, estrogen and levormeloxifene that caused fulldisappearance of epithelium atrophy, maintained endometrium histology atthe levels of sham controls.

[0373] Compound I and II of the invention respectively administered at0.1, 1 and 10 mg/kg/day and 0.1 and 1 mg/kg/day for 4 weeks by oralroute, significantly reduced bone loss while lowering serum cholesterollevels.

[0374] These protective effects were achieved at a dose with minimaluterine stimulation.

[0375] Taken together, the reported results show that the benzopyranderivatives of the invention are provided with an interesting SERM(Selective Estrogen Receptor Modulation) activity, comparing favorablyin tissue selectivity with the structurally related reference compoundsof the prior art.

[0376] In Vitro Study—MCF-7 Cell Proliferation

[0377] In this test the Compound I has been compared with the twomarketed products, raloxifene and tamoxifene.

[0378] The tumor cells were obtained from American Type CultureCollection (ATCC HTB-22). The culture media used was Minimum Essentialmedium with 10% fetal bovine serum, and media were supplemented with 1%antibiotic-antimycotic. Aliquots of 100 μl of cell suspension(5×103/well) were placed in 96-well microtiter plates in an atmosphereof 5% CO2 at 37° C. After 24 hours, 100 μl of growth medium, 2 μl oftest solution, mitomycin or vehicle (DMSO, final concentration 0.4%),was added respectively per well in duplicate for an additional 72-hoursincubation. At the end of incubation, 20 μl of alamarBlue 75% reagentwas added to each well for another 6-hour incubation before detection ofcell viability by fluorescent intensity (excitation at 530 nm andemission at 590 nm).

[0379] The Compound I and two comparison compounds, raloxifene andtamoxifene exhibited significant growth inhibition relative to thevehicle treated control group at concentrations between 1 and 100 μM.Further, under the conditions of this experiment, tamoxifene and theCompound I were more potent (Inhibitory Concentration IC₅₀=6.0 and 6.5μM, respectively) than raloxifene (IC₅₀=11 μM).

[0380] Total growth inhibition was observed at 11 μM for tamoxifene andthe Compound I or 18 μM for raloxifene, whereas the 50% lethalconcentrations (LC50) were 22 (tamoxifene), 20 (Compound I) and 29(raloxifene) μM.

[0381] The reported results confirm that the compounds of the inventionact as a potent and selective tissue-specific estrogen agonist andantagonist.

[0382] The tested compounds have a better pharmacological profile thanthe structurally related reference compound benzopyran derivativelevormeloxifene and compare favourably with raloxifene, one of the firstSERMs developed for clinical use.

[0383] In view of their therapeutic use, the compounds of the inventioncan be opportunely combined with the usual pharmaceutically acceptableexcipients for the preparation of pharmaceutical compositions forparenteral, oral, nasal, rectal, subdermal or transdermal administrationaccording to conventional methods.

[0384] One skilled in this art may formulate the compounds in anappropriate manner, and in accordance with accepted practices, such asthose disclosed in Remington's Pharmaceutical Sciences, Gennaro ed.,Mack Publishing Co., Easton, Pa. 1990.

1. 2H-1-Benzopyran derivatives of general formula:

wherein R₁ and R₂ are independently H, alkyl, haloalkyl, alkenyl orhaloalkenyl, or, when they are alkyl, together with nitrogen atom theyare bound to, they can form a 4- to 8-membered heterocyclic ring X is H,alkyl, aryl, nitro, halo, O—R₃ wherein R₃ is H, alkyl, aryl, alkanoyl,aryloyl X₁ is H, alkyl or alkoxy and when X and X₁ are alkyl they canform, together with the carbon atom they are bound to, a fused aromaticring to give an α-naphthalenyl Y is H, alkyl, alkanoyl, aryloyl,alkylaminocarbonyl, alkyloxycarbonyl Z is H, O—R₄ wherein R₄ is H,alkyl, alkanoyl, aryloyl m is 1,2 n is 0,1 p is 2-6

represents a single or double bond between the atoms in positions 3-4 or4-4a: when n is 1 the double bond may be alternatively exocyclic orendocyclic to give respectively a 4-benzylidenechroman for m=1 or a4-benzyl-chrom-3-ene for m=2; when n is 0 the endocyclic bond 3-4 may bea single or a double bond and when n is 0 and the endocyclic bond 3-4 isa single bond, Z is not H, and the pharmaceutically acceptable saltsthereof.
 2. 2H-1-Benzopyran derivatives according to claim 1 wherein: R₁and R₂ are alkyl and together with nitrogen atom to which they are boundform a piperidine ring, X is H or C₁-C₄ alkoxy, X₁ is H, Y is H, C₁-C₅alkanoyl or aryloyl Z is H, m is 2, n is 1, p is 2 the bond between theatoms in position 3-4 is a double bond and the bond between the atoms inposition 4-4a is a single bond.
 3. The compound according to claims 1-2that is3-phenyl-4-(4-(2-(N-piperidinyl)ethoxy)benzyl)-7-hydroxychrom-3-ene, itsesters and salts.
 4. The compound according to claims 1-2 that is3-(4-methoxy)phenyl-4-[[4-[2-(1-piperidinyl)ethoxy]phenyl]-methyl]-2H-1-benzopyran-7-ol,its esters and salts.
 5. Pharmaceutical compositions containing as theactive ingredient at least one compound as claimed in claims 1-4together with pharmaceutically acceptable excipients.
 6. The use of thecompounds according to claims 1-4 for the preparation of a medicamentuseful in the prevention and treatment of a number of postmenopausalpathologies, in particular osteoporosis, coronary heart disease andestrogen dependent human cancer.